Báo cáo khóa học: Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4 ppt

Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4 Miglena E. Stefanova 1 , Joshua Tomberg 2 , Christopher Davies 3 , Robert A. Nicholas 2 and William G. Gutheil 1 1 Division of Pharmaceutical Sciences, University of Missouri-Kansas City, USA; 2 Department of Pharmacology, University of North Carolina at Chapel Hill, North Carolina, USA; 3 Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina, USA The penicillin-binding proteins (PBPs) are ubiquitous bac- terial enzymes involved in cell wall biosynthesis, and are the targets of the b-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for b-lactam binding, DD -carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 M )1 Æs )1 ). Against a setoffivea-ande-substituted L -Lys- D -Ala- D -Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N e -acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N e -Cbz group demonstrated pro- nounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k 2 (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site- directed agents, N-chlorosuccinimide, elastinal, iodoaceta- mide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac 2 - L -Lys- D -Ala- D -Ala (k cat /K m ) was bell-shaped, with pK a values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other DD -carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs. Keywords: DD -carboxypeptidase; penicillin-binding protein. Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes that catalyze the last steps in cell wall biosynthesis (reviewed in [1–5]), and are the targets of the b-lactam antibiotics. In Gram-negative bacteria these enzymes cata- lyze the reactions shown in Scheme 1. Each bacterial species has a number of PBPs; for example, Escherichia coli has eight classically known PBPs, labeled 1A, 1B, and 2–7, as well as several recent additions including PBP 1C [6] and PBP 6b [7]. PBPs have molecular masses of 20–120 kDa and can be divided into two groups, the low molecular mass (LMM) PBPs and the high molecular mass (HMM) PBPs [3]. The LMM PBPs have a transpeptidase/hydrolase domain whereas the HMM PBPs possess an additional domain N-terminal to the PBP domain, which in some cases catalyze a penicillin-insensitive transglycosylase reaction. Different PBPs have different propensities for hydrolysis and/or transpeptidase reactions [5]. HMM PBPs are often the lethal targets for b-lactam antibiotics, whereas LMM PBPs are not lethal targets. Two LMM PBPs have, however, been found to play important roles in cell division and cell shape; PBP 3 from Streptococcus pneumoniae is required for normal septum formation [8], and PBP 5 from E. coli is essential for normal cell shape [9]. Neisseria gonorrhoeae has only three PBPs visible on gels when [ 3 H]penicillin G-labeled membranes are analyzed by SDS/PAGE and fluorography (PBPs 1, 2, and 3) [10]. Analysis of the recently completed genome sequences of N. meningitidis [11] and N. gonorrhoeae (GenBank acces- sion number AE004969) revealed a fourth gonococcal PBP, termed PBP 4 (GenBank accession number AF156692). PBPs 1 and 2 are HMM PBPs and are the major antibiotic killing targets for N. gonorrhoeae [10]. PBP 1 is the gono- coccal homologue of E. coli PBP 1A and likely catalyzes both glycan polymerization and transpeptidation during cell elongation [12]. PBP 2 is the homologue of E. coli PBP 3 and likely functions during cell division [13]. PBPs 3 and 4 are LMM PBPs, which are not lethal targets, and their role in cell wall biosynthesis is unknown. In a recently completed study, PBP 3 was found to be an exceptionally active carboxypeptidase, and exhibited high rates of acylation by Correspondence to W. G. Gutheil, Division of Pharmaceutical Sciences, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, Missouri 64110. Fax: +1 816 235 5190, Tel.: +1 816 235 2424, E-mail: gutheilw@umkc.edu Abbreviations: Alk-BSA, alkylated BSA; AR, Amplex TM Red; Boc, tert-butoxycarbonyl; Caps, 3-(cyclohexyl amino)-1-propane sulfonic acid; Cbz, carbobenzyloxy; CPase, carboxypeptidase; EC, Escherichia coli; HMM, high molecular mass; LMM, low molecular mass; NG, Neisseria gonorrhoeae;OPD,o-phenylenediamine; PBP, penicillin- binding protein; KAA, L -Lys- D -Ala- D -Ala; TEA, triethylamine. (Received 23 September 2003, accepted 21 October 2003) Eur. J. Biochem. 271, 23–32 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03886.x b-lactam antibiotics. This study also found that deletion of PBP 3 and PBP 4 individually had only a slight (P >0.05, not statistically significant) effect on the growth and viability of N. gonorrhoeae, whereas deletion of both resulted in a modest (P < 0.05, statistically significant) decrease in rate of cell growth. Moreover, scanning electron micrographs of these cells revealed a change in morphology in cells lacking both PBP 3 and PBP 4, but not in cells lacking only one of these enzymes, suggesting a role for both enzymes in normal cell wall biosynthesis [13a]. Characterization of PBPs in terms of enzymatic proper- ties, structure/function relationships, and catalytic mechan- ism [14,15] provides useful information for understanding the role of individual PBPs in bacterial cell wall biosynthesis, as well as a basis for the development of new inhibitors for the PBPs [16] that could lead to the development of new antibacterial agents. Enzymatic characterization efforts on the PBPs have focused on LMM PBPs since in general only LMM PBPs give detectable enzymatic activity, for reasons which are as yet unclear. There are a number of similarities between LMM and HMM PBPs in terms of active site sequence [3] and architecture [14,17,18]. Both groups of enzymes also act on cell wall peptidoglycan as their substrate, and are reactive with b-lactam antibiotics. Detailed studies of the LMM PBPs are therefore expected to provide important information on the enzymological properties of the PBP class of enzymes as a whole, and information on the as yet unexplained functional and catalytic differences between the LMM and HMM PBPs. The present study reports the cloning, expression, puri- fication, and enzymological characterization of N. gonor- rhoeae (NG) PBP 4. NG PBP 4 was examined for b-lactam binding, DD -carboxypeptidase activity, substrate specificity, transpeptidase activity, sensitivity to general enzyme inhibit- ors, and pH dependence. Comparison of the enzymatic properties of NG PBP 4 with other DD -carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechan- istic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs. Experimental procedures General materials and reagents Tris, D -Ala, horseradish peroxidase (Type X; 21 mgÆmL )1 as an ammonium sulfate suspension; 250 UÆmg )1 ), ampi- cillin, FAD, o-phenylenediamine (OPD), diacetyl- L -Lys- D -Ala- D -Lac (Ac 2 -KA- D -Lac) and D -lactate dehydrogenase were from Sigma Chemical Co. Pig kidney D -amino acid oxidase (6.0 mgÆmL )1 as an ammonium sulfate suspension; 12 UÆmg )1 ) was from Roche Molecular Biochemicals. The PBP/ DD -carboxypeptidase substrate diacetyl- L -Lys- D -Ala- D -Ala (Ac 2 -KAA) and substituted XY-KAA substrates were synthesized using standard methods of solution phase peptide synthesis [19,20]. Amplex TM Red (10-acetyl-3,7- dihydroxyphenoxazine; AR) was from Molecular Probes and the QuantaBlu TM substrate solution was from Pierce Chemical Co. Protein content was determined by using the Micro Bradford assay (Sigma) according to the manufac- ture’s protocol. Alkylated BSA (Alk-BSA) was prepared as described previously [15]. Cloning, expression and protein purification The NG PBP 4 coding sequence was amplified from N. gonorrhoeae strain FA1090 genomic DNA with primers based on the GenBank sequence (accession number AF156692). The up primer annealed to codons 29–36 inclusive and contained an in-frame BamHI site at its 5¢-end, whereas the down primer annealed to the last seven codons of the coding sequence and contained an EcoRI site at its 5¢-end. The amplified fragment was cloned into a modified pMAL-C2 vector (New England Biolabs), which fused PBP 4 with hexahistidine-tagged maltose-binding protein via a linker sequence containing a cleavage site for Tobacco Etch Virus protease. The sequence of the cloned insert was identical to the sequence both from the GenBank entry for NG PBP 4 and from the completed FA1090 genome (accession numbers AF156692 and AE004969, respectively). The fusion protein was induced with 0.1 m M isopropyl- thio-b- D -galactoside in E. coli MC1061 cells and purified from cell lysates on a nickel chelating column. The purified fusion protein was cleaved with Tobacco Etch Virus protease, and the digest was repurified on a nickel chelating column. PBP 4 did not elute in the flow-through, but instead eluted from the column in 15 m M imidazole, while maltose-binding protein and uncleaved fusion protein eluted Scheme 1. PBP-catalyzed transpeptidase and carboxypeptidase reac- tions in E. c oli. 24 M. E. Stefanova et al. (Eur. J. Biochem. 271) Ó FEBS 2003 in 150 m M imidazole. Puried PBP 4 was estimated to be > 95% pure by SDS/PAGE, and was stored at )80 C. Determination of the acylation rate of NG PBP 4 by several b-lactam antibiotics The reaction mechanism for the interaction of PBPs with peptide and b-lactam antibiotics is: E ỵ S ! K 0 ES ! k 2 E S ! k 3 E ỵ P 1ị The constant k 2 /KÂ, which describes the formation of the covalent acyl-enzyme complex at low (subsaturating) con- centrations of b-lactams, was determined from time courses with [ 14 C]penicillin G essentially as described [21]. NG PBP 4 (48 lg, 1.2 nmol) was diluted into 150 lL binding buffer (50 m M sodium phosphate pH 7.0, 10% glycerol) and mixed with an equal volume of 100 l M [ 14 C]penicillin G in binding buffer. At timed intervals, 20-lL aliquots were removed, mixedwith5 mL5%trichloroaceticacid(w/v),andincubated on ice for 15 min. The acidied proteins were passed through #30 glass ber lters (Schleicher and Schuell) and the lters were washedtwice with5 mLeachof 1%trichloroacetic acid/ 33% methanol. The lters were then air dried, placed in scintillation vials with 3 mL Scinti-safe scintillation uid (Fisher Scientic), and counted. k 2 /K constants for ampicillin and ceftriaxone were determined by the competition method against [ 14 C]peni- cillin G [21]. A xed concentration of [ 14 C]penicillin G of 0.5 l M and concentrations of the unlabeled antibiotics that inhibited binding by % 50% were incubated with PBP 4 in sodium phosphate buffer pH 7, at room temperature for 3 min and the level of radioactivity bound to the proteins was quantied as described above. Equation (2) was used to calculate k 2 /K constants, where EC 0 and EC U represent the amount of acyl-enzyme complex formed in the absence and presence of the unlabeled antibiotic, respectively, and C U and C L are the concentrations of the unlabeled and labeled antibiotic, respectively. k 2 K 0 U ẳ k 2 K 0 L EC 0 EC U ịC L EC U ịC U 2ị Enzyme activity assays $ D -Ala- D -Ala carboxypeptidase (CPase) activity was deter- mined by uorescence-based detection of D -Ala in micro- titer plate-based assays as described in detail previously [15,22]. Assays (50 lL) were performed in 100 m M pyro- phosphate, 100 m M NaCl, 0.5 mgặmL )1 Alk-BSA, at pH 8.5. PBP 4 was diluted in the same buffer, and added to reactions to start assays. Activity against the depsipeptide (ester) substrate Ac 2 -KA- D -Lac was determined by detec- tion of D -Lac using D -lactate dehydrogenase as described previously [23], but with assays performed in microtiter plates and the NADH product measured uorimetrically (excitation at 325 nm, emission at 465 nm). D -Lac was used as a standard. Control experiments were performed with PBP 4 in the absence of substrate, and substrate in the absence of PBP 4. In the case of the D -Lac based substrate which has a labile ester bond, a low level of esterolysis was observed in control experiments minus NG PBP 4, which was subtracted from experimental values plus NG PBP 4. The linearity of NG PBP 4 catalyzed reactions was veried in a time course experiment. NG PBP 4 (105 n M ) wasaddedto10m M (subsaturating) Ac 2 -KAA in the standard CPase assay mixture. Reactions were stopped at various times by the addition of ampicillin to 50 lgặmL )1 (135 l M ) and the accumulation of the hydrolysis product ( D -Ala) was determined uorimetrically. No product ( D - Ala) was produced for the zero time point demonstrating that this ampicillin concentration completely blocked PBP 4 activity. Additional preliminary experiments further dem- onstrated that the apparent K i values for ampicillin and penicillin G are in the low nanomolar range (data not shown). Transpeptidase assays PBPs can catalyze hydrolysis and/or transpeptidase reac- tions (Scheme 1). To determine the ability of NG PBP 4 to catalyze transpeptidase reactions, a model transpeptidase reaction was performed with 10 m M Ac 2 -KAA as the acyl group donor and variable concentrations of glycine as the acyl group acceptor [24,25]. NG PBP 4 was added to 175 n M and reactions run for 150 min before stopping by addition of ampicillin. This relatively high NG PBP 4 concentration was sufcient to convert 5% of substrate to products, which allowed accurate product determination by HPLC. The large amount of D -Ala hydrolysis product produced at this level of turnover was determined using low sensitivity OPD-based microtiter plate assays described previously [23,26]. Transpeptidase (Ac 2 -KAG) and hydro- lysis (Ac 2 -KA) products were quantied by reverse-phase HPLC on a C18 column (5 lm, 0.46 ã 25 cm) with a water/ acetonitrile gradient. The column was equilibrated in 100% A. Gradient: 025% B in 15 min [A: 0.1% (v/v) triuoro- acetic acid in water; C: 0.09% (v/v) triuoroacetic acid in acetonitrile;B:30%A/70%C(v/v)]. Substrate specicity L -Lys- D -Ala- D -Ala (KAA) based substrates with various N a (X) and N e (Y) substituents were incubated with NG PBP 4 at increasing substrate concentrations (050 m M XY- KAA). Data were analyzed by tting with the appropriate equations by nonlinear regression using BMDP statistical software (SPSS Science). Effect of inhibitors and reagents Inhibitors and reagents at 1 m M were incubated with the enzyme (21 n M )for1hat25Cinanassaymixture containing all components of the CPase assay except for the substrate. Ac 2 -KAA was then added to the assay. Reactions were stopped by the addition of developing reagent containing AR and ampicillin as described above. The activity of untreated enzyme was taken as 100%. pH dependence and pH stability The effect of pH on NG PBP 4 activity was studied and data analyzed as described in detail previously [15]. Activity ể FEBS 2003 N. gonorrhoeae PBP 4 (Eur. J. Biochem. 271)25 was assayed in a series of overlapping buffers at 50 m M buffer, 100 m M NaCl, 0.5 mgÆmL )1 Alk-BSA pH 3.5– 12.25, with 10 m M Ac 2 -KAA as the substrate. A control study was performed to determine the effect of pH on NG PBP 4 stability, also as described previously [15]. Amino acid sequences The amino acid sequences used in this study were from the Swiss-Prot database. The accession numbers were: NG PBP 3, O85665; NG PBP 4, Q9XBT7; E. coli (EC) PBP 5, P04287; Streptomyces K15 PBP, P39042; TEM-1 b-lactamase, P00810. Results b-Lactam binding activity The gene encoding NG PBP 4 was amplified from N. gon- orrhoeae FA1090 DNA, expressed in E. coli and purified as described in Experimental procedures. To verify that the purified protein was indeed a PBP, we carried out penicillin- binding assays with [ 14 C]penicillin G. As shown in Table 1, NG PBP 4 displayed a k 2 /K¢ acylation rate constant of 30 000 M )1 Æs )1 with [ 14 C]penicillin G, and an even higher constant (56 000 M )1 Æs )1 ) with ceftriaxone. In addition to these assays, NG PBP 4 activity was found to be completely inhibited by 140 l M ampicillin used to stop DD -CPase assays, and an apparent K i in the low nanomolar range was observed in additional control experiments (data not shown). Substrate specificity Most LMM PBPs are active as DD -carboxypeptidases. NG PBP 4 was characterized against several $ D -Ala- D -Ala based peptide substrates, as summarized in Fig. 1 and Table 2. For data following expected Michaelis–Menten behavior nonlinear regression with the form of the Micha- elis–Menten equation shown in Eqn (3) was used to obtain values and standard errors (SE) for k cat and K m , and the form of this equation shown in Eqn (4) to obtain the value and SE for k cat /K m . v=E T ¼ k cat ½S K m þ½S ð3Þ v=E T ¼ ðk cat =K m ÞÂK m ½S K m þ½S ð4Þ In the case of Boc-Cbz-KAA and Ac-Cbz-KAA substantial substrate inhibition was observed (Fig. 1). In these two cases, only the data points from 0 to peak activity were Fig. 1. Substrate specificity of NG PBP 4. CPase activity was assayed as described in the text using 35 n M NG PBP 4, and a reaction time of 90 min. D -Ala product was detected with QuantaBlu TM assays. j, Boc-Cbz-KAA; r, Boc-Ac-KAA; s, Ac-C-KAA; n, Ac 2 -KAA; h, Boc-H-KAA; d, Ac 2 -KA- D -Lac. (A) High activity range showing activity against Ac 2 -KA- D -Lac. (B) Middle activity range. (C) Low activity range. Table 1. k 2 /K¢ values for b-lactam antibiotic binding to NG PBP 4. Standard errors are given in parentheses. b-Lactam antibiotic k 2 /K¢ ( M )1 Æs )1 ) [ 14 C]Penicillin G 30 000 (2000) Ampicillin 3800 (300) Ceftriaxone 56 000 (3000) 26 M. E. Stefanova et al. (Eur. J. Biochem. 271) Ó FEBS 2003 included in the statistical analysis using Eqn (4). The k cat /K m values, which reflect the activity of the enzyme at subsat- urating (low) substrate concentrations, were determined using this analysis and are accurate. However, the apparent K m values will be lower than the true K m values, as will the k cat values. An effort to fit the full concentration profile to several substrate inhibition models was attempted, including noncompetitive and uncompetitive substrate inhibition models. An uncompetitive model [Eqn (5), where K is is the substrate inhibition constant] gave the best fit of experimental data, but the standard errors for kinetic parameters were high due to overlapping K is and K m values. The k cat and K m values for these two substrates could therefore not be resolved, and are not reported. v=E T ¼ k cat ½S K m þ½Sþ½S 2 =K is ð5Þ Values for k cat and K m for Boc-Ac-KAA and Boc-H-KAA also could not be obtained, in these cases due to the lack of apparent substrate saturation. Since the possibility of substrate inhibition also exists for the other two substrates, Ac 2 -KAA and Ac 2 -KA- D -Lac, the values for k cat and K m reported in Table 2 are given as the apparent values, and are the minimum values for these parameters ) the true values could be higher. Values for kinetic parameters are also based on the assumption that all enzyme is catalytically active. NG PBP 4 showed highest activity (k cat /K m )withN a - acetylated substrates. Replacement of the N a -acetyl group with the bulky Boc group decreased activity. Lowest activity was obtained for Boc-H-KAA, which has a free (unacyl- ated) N e . NG PBP 4 demonstrated an order of magnitude higher activity against Ac 2 -KA- D -Lac (1730 M )1 Æs )1 )than against the analogous peptide substrate Ac 2 -KAA (76 M )1 Æs )1 ), with apparent values for K m essentially the same (60 m M vs. 40 m M , respectively). Transpeptidase activity No transpeptidase product was observed for NG PBP 4 catalyzed transpeptidation reactions with glycine as the transpeptidase acceptor and Ac 2 -KAA as the acyl group donor. A constant amount of accumulated D -Ala with increasing Gly concentration was also observed (data not shown). These results demonstrate that NG PBP 4 does not show transpeptidase activity in a model transpeptidase reaction between Ac 2 -KAA and Gly. pH optimum and stability The pH dependence of k cat /K m for NG PBP 4 catalyzed hydrolysis of Ac 2 -KAA is presented in Fig. 2A. NG PBP 4 gave a bell-shaped pH profile with an optimum in the range 7.5–9.0. Data from TEA buffers demonstrated anomalous behavior and were excluded from the analysis for pK a values. The k cat /K m vs. pH data was analyzed to determine the pK a values as described previously [15]. pK a values were 6.9 (SE ¼ 0.1) and 10.1 (0.1). No anomalous effect of buffers other than TEA was observed, except for a slight preference for Caps over carbonate buffers. NG PBP 4 was fully stable at pH 5.25–12.25 for 60 min at 25 °C (Fig. 2B). Fig. 2. Effect of pH on activity and stability of NG PBP 4. (A) pH-rate (k cat /K m ) profile for NG PBP 4 hydrolysis of Ac 2 -KAA. The NG PBP 4 concentration was 21 n M , and the reaction time was 75 min. D -Ala was detected with AR based assays. n, Citrate; s, Pi; r, PPi; e, TEA; d, CO 3 2– ; h, Caps. The solid line represents the best fit curve calcu- lated as described in Materials and methods with values of pK 1 ¼ 6.9 (0.1) and pK 2 ¼ 10.1 (0.1). (B) pH stability profile of NG PBP 4. Experimental conditions were as described above. Symbols are as in A. Table 2. Kinetic constants for hydrolysis of D -Ala and D -Lac based substrates by NG PBP 4. Standard errors are given in parentheses. H, No substituent; KAA, L -Lys- D -Ala- D -Ala; NS, not available due to lack of apparent substrate saturation. SI, not available due to substrate inhibition. Substrate a k cat /K m ( M )1 Æs )1 ) K m (m M ) b k cat (s )1 ) b Ac 2 -KAA 76 (2) 40 (2) 3.1 (0.1) Ac-Cbz-KAA 60 (10) SI SI Boc-Cbz-KAA 27 (4) SI SI Boc-Ac-KAA 11.3 (0.2) NS NS Boc-H-KAA 3.58 (0.04) NS NS Ac 2 -KA- D -Lac 1730 (70) 60 (6) 100 (6) a The N a -substituent of Lys is given first, N e -substituent is given second. b Minimum apparent value as discussed in text. Ó FEBS 2003 N. gonorrhoeae PBP 4 (Eur. J. Biochem. 271)27 Effect of enzyme inhibitors and reagents NG PBP 4 was fully inhibited by the oxidizing agent N-chlorosuccinimide (Table 3). Cysteine modifying rea- gents iodoacetamide and iodoacetic acid, and the arginine modifying reagent phenylglyoxal significantly inhibited enzyme activity (40–50%). Among serine protease inhibi- tors only elastinal gave substantial inhibition, although methyl boronic acid showed modest inhibition. Serine- directed organic phosphates were ineffective as inhibitors. None of the tested metal ions or chelators significantly affected enzyme activity. Discussion NG PBP 4 was identified from the complete genomic sequence of N. gonorrhoeae. Although NG PBP 4 is not visible when [ 3 H]penicillin G-labeled membranes are analyzed by SDS/PAGE and fluorography [10], a significant decrease in growth rate and accompanying morphological abnormalities occurred only when both PBP 3 (the other LMM PBP in N. gonorrhoeae) and PBP 4 were deleted [13a], strongly suggesting that PBP 4 plays a role in cell wall synthesis. In this study NG PBP 4 was cloned, overex- pressed, purified, and characterized to provide data required for mechanistic and structure–function correlations with other PBPs, and to provide a basis for understanding the possible physiological function of this enzyme. As NG PBP 4 was not observed in [ 3 H]penicillin G labeled membranes [10], it was possible that this protein does not bind b-lactam antibiotics. However, b-lactam binding experiments demonstrated that NG PBP 4 does bind [ 14 C]penicillin G and several other b-lactam antibiotics with reasonably high k 2 /K¢ acylation rate constants (Table 1). Although the k 2 /K¢ value of [ 14 C]penicillin G with NG PBP 4 is lower than that determined for NG PBP 3 (198 000 M )1 Æs )1 ), it is considerably higher than that of E. coli PBP 5, which has a k 2 /K¢ for [ 14 C]penicillin G of 390 M )1 Æs )1 [26a]. Moreover, NG PBP 4 was completely inhibited by ampicillin at the 140 l M concentration used to stop carboxypeptidase reactions (data not shown). The lack of an observable band corresponding to NG PBP 4 in [ 3 H]penicillin G-labeled gonococcal membranes following SDS/PAGE and fluorography therefore cannot be due to a Table 3. Effect of general enzyme inhibitors and reagents on the carboxypeptidase activity of three LMM PBPs. NG PBP 4 data, this paper; NG PBP 3 data, unpublished observations; EC PBP 5 data, [15]. Inhibitor/reagent Residual activity (%) SpecificityNG PBP 4 NG PBP 3 EC PBP 5 Blank (untreated enzyme) 100 100 100 PMSF 104 101 113 Ser protease Methanesulfonyl fluoride 96 100 82 Ser protease DIFP 103 100 78 Ser protease Leupeptin 89 99 108 Ser, Cys protease Elastinal 45 100 66 Ser protease Boric acid 85 99 89 Ser protease Phenylboronic acid 89 24 73 Ser protease Methylboronic acid 76 92 100 Ser protease pHMB 81 97 0 Cys Tetranitromethane 106 93 100 Tyr, Cys N-Ethylmaleimide 91 99 9 Cys Iodoacetamide 56 98 83 Cys Iodoacetic acid 59 101 96 Lys, Cys N-Chlorosuccinimide 8 5 44 Met, Cys, Trp Formaldehyde 95 104 44 Cys, Lys, His Acetic anhydride 104 103 74 Lys Methylacetimidate 105 97 95 Lys Diethyl pyrocarbonate 109 104 90 Lys, His, Tyr Ethyl trifluorothioacetate 104 102 91 Lys EDTA 115 102 96 Metal chelator 1,10-Phenanthroline 102 97 96 Metal chelator Phosphoramidon 100 105 99 Zn protease 2-Aminoethylphosphonic acid 99 103 96 alanine analog Phenylglyoxal 47 79 109 Arg ZnCl 2 104 103 97 CdCl 2 99 105 91 CaCl 2 107 105 87 CoCl 2 103 103 94 CuCl 2 92 104 95 MnCl 2 89 105 100 MgCl 2 99 106 97 28 M. E. Stefanova et al. (Eur. J. Biochem. 271) Ó FEBS 2003 lack of interaction with penicillin G, and suggests that N. gonorrhoeae grown in culture expresses only a low level of this protein. Unidentified PBPs with a role in cell wall morphology have previously been observed in E. coli [27]. NG PBP 4 activity against the $ D -Ala- D -Ala substrate Ac 2 -KAA was fairly typical for a PBP with a k cat value of 3s )1 , K m value of 3 m M , and k cat /K m of 80 M )1 Æs )1 (Table 2). NG PBP 4 demonstrated substantial variation in its activity against a set of XY- L -Lys- D -Ala- D -Ala peptide substrates and the depsipeptide substrate Ac 2 - L -Lys- D -Ala- D -Lac (Table 2, Fig. 1). For substituents on the N e of Lys, Ac- or Cbz-substituted substrates gave highest activity whereas the absence of either an Ac or Cbz group in Boc-H- KAA was associated with lowest activity, indicating a preference of NG PBP 4 for N e -acylated substrates. For comparison (Table 4), NG PBP 3 demonstrated a similar preference for N e -acylated substrates, but in contrast was more active with bulky substituents on the N a -position, and EC PBP 5 demonstrated a notable lack of significant preference for any of these derivatives. The observation that NG PBP 3 and NG PBP 4 show a preference for N e - substituted substrates suggests that the preferred substrates for both LMM neisserial PBPs would be crosslinked pentapeptides in the cell wall. PBP-catalyzed reactions proceed through an acyl-enzyme intermediate (Scheme 2). Depsipeptide (peptide-ester) sub- strates for the PBPs often show a large increase in reaction rate over the homologous amide substrates [28]. Since both the depsipeptide (Ac 2 - L -Lys- D -Ala- D -Lac) and amide (Ac 2 - L -Lys- D -Ala- D -Ala) substrates proceed through the same acyl-enzyme intermediate (E-S in Scheme 2), k 3 will be the same for both substrates. A greater turnover number (k cat ) for a depsipeptide substrate is therefore attributed to a higher k 2 , and is an indication that k 2 (acylation) is the rate determining step for hydrolysis of the peptide substrate [28,29]. Conversely, if peptide and depsipeptide substrates show similar turnover numbers then this is evidence that k 3 (deacylation) is rate determining. NG PBP 4 demonstrated a much higher k cat (30-fold) against Ac 2 - L -Lys- D -Ala- D -Lac than against Ac 2 - L -Lys- D -Ala- D -Ala (Ac 2 -KAA) (Table 2), evidence that k 2 is rate determining for NG PBP 4-catalyzed hydrolysis of Ac 2 -KAA. Classic studies using this approach demonstrated that k 2 is also rate determining for EC PBP 5-catalyzed hydrolysis of Ac 2 -KAA [28]. In contrast, deacylation (k 3 ) appears rate limiting for both NG PBP 3 [13a] and S. aureus PBP 4 [28,30] catalyzed hydrolysis of amide and ester substrates. A number of active site-directed inhibitors and metal ions were tested for their ability to inhibit NG PBP 4 (Table 3). N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. As there are no cysteines in NG PBP 4, inhibition by iodoacetamide and iodoacetic acid is attributed to lysine modification. Com- parison with inhibitor data from NG PBP 3 and EC PBP 5 (Table 3) demonstrates that all three enzymes were largely unaffected by general inhibitors of serine proteases, which is noteworthy given the role of a serine acyl-enzyme interme- diate in PBP catalysis [28]. This result is also consistent with previous observations with the Streptomyces R61 carb- oxypeptidase [31]. The only inhibitor effective against all three enzymes was the oxidizing agent N-chlorosuccinimide whichinhibitedfullyNGPBP4andNGPBP3,andtoa substantial degree EC PBP 5 (Table 3). Thus, the PBPs appear notably resistant to most active site-directed rea- gents. In contrast, the PBPs appear generally sensitive to transition state analogs such as peptide boronic acids [16] and peptide phosphonates [32]. pH dependence is a fundamental enzyme characteristic relevant to physiological, mechanistic, and kinetic under- standing of enzyme catalyzed reactions [33]. The physio- logical significance of pH dependence is of particular relevance for the PBPs and the b-lactamases, which are exposed to the extracellular environment. pH dependence of N. gonorrhoeae growth and cell wall biosynthesis has been investigated ([34] and references therein). N. gonorrhoeae grows best at pH 7.2, with a growth window of 5.8–8.4. There is a significant difference in N. gonorrhoeae growth environment between the infected female and male, with the vagina having a pH range of 4.2–7.5 and the male urethra having a pH of 6.2–8.4. The pH dependence of NG PBP 4 (pK a values of 6.9 and 10.1) therefore overlaps the alkaline side of the physiological growth range of N. gonorrhoeae in both males and females, with better overlap with male growth conditions. The pH profiles for NG PBP 3 and 4 are relatively acidic compared to that of EC PBP 5, which further highlights the contrast between the pH profile of EC PBP 5 (pK a values 8.2 and 11.1) and the physiological growth range of E. coli (pH 6–8) [15]. This is especially notable as EC PBP 5 is required for normal cell shape [9]. The pK a values for NG PBP 4 of 6.9 and 10.1 are almost identical to the pK a values for NG PBP 3 of 6.8 and 9.8 [13a]. The pH profile of NG PBP 4, especially the acidic limb, is also similar to the pH profile for the Class A TEM b-lactamase with pK a values of 5.0–6.2 for the acidic limb and 8.5 for the basic limb [35,36] (Table 5). The conserved W-loop in Class A b-lactamases contains a highly conserved Glu residue (Glu166) which is believed to act as the general Scheme 2. PBP-catalyzed substrate hydrolysis. Table 4. Enzyme activities (k cat /K m ) for hydrolysis of D -Ala and D -Lac based substrates by NG PBP 3, NG PBP 4 and EC PBP 5. NG PBP 4 data, this paper; NG PBP 3 data, [13a]; EC PBP 5 data, [15]. Stan- dard errors are shown in parentheses. Substrate Enzyme activity ( M )1 Æs )1 ) NG PBP 4 NG PBP 3 EC PBP 5 Ac 2 -KAA 76 (2) 29 000 (2000) 12 (1) Ac-Cbz-KAA 60 (10) 142 000 (6000) 12 (1) Boc-Cbz-KAA 27 (4) 180 000 (30 000) 21 (1) Boc-Ac-KAA 11.3 (0.2) 62 000 (6000) 9 (1) Boc-H-KAA 3.58 (0.04) 8300 (400) 11 (1) Ac 2 -KA- D -Lac 1730 (70) 12 300 (800) 700 (10) a a [23]. Ó FEBS 2003 N. gonorrhoeae PBP 4 (Eur. J. Biochem. 271)29 base in the deacylation step of the catalytic cycle [37,38], and it is considered responsible for the acidic limb (pK a 5–6) in the pH profile [35]. In contrast, EC PBP 5 contains an active site loop spatially equivalent to the W-loop in b-lactamases (W-like loop) but which lacks a suitably positioned Glu residue [14]. Although His151 on the W-like loop of EC PBP 5 aligns with Glu166 in TEM b-lactamase, this residue does not appear properly oriented to participate in catalysis [14] and the pK a values for EC PBP 5 carboxyp- eptidase activity of 8.2 and 11.1 were assigned to Lys47 (SXXK motif) and Lys213 (KTG motif), respectively [15]. NG PBP 4 and NG PBP 3 are both Class C LMM PBPs, for which no crystal structures are currently available. At the sequence level, Class C LMM PBPs are most similar to the Class A b-lactamases, followed by Class A PBPs such as EC PBP 5 and the Streptomyces K15 enzyme, followed by a relatively remote relationship to the Class B LMM PBPs such as the Streptomyces R61 enzyme [39]. Both gonococcal enzymes are carboxypeptid- ases, both have the highly conserved active site motifs common to most members of this class of enzymes, and both show a readily identifiable W-like loop motif in sequence alignments (Table 6). However, assigning pK a values to specific residues in NG PBP 4 and NG PBP 3 is difficult, especially in the absence of crystal structures. By analogy with TEM b-lactamase and EC PBP 5, the basic pK a of NG PBP 4 can be reasonably assigned to the KTG motif lysine (Lys261) (Tables 5 and 6). The acidic limb of NG PBP 4 at pH 6.9 is however, less easy to assign to Lys102 and, at first glance, a residue with a more acidic pK a would seem more appropriate. One such candidate is Glu195, which is present on the W-like loop (Table 6). The presence of a conserved Gly-Leu/Ile motif at the heart of the W-like loop suggests that W-like loops may exhibit similar structures within classes A and C of the LMM PBPs. Indeed the main chain atoms of those for EC PBP 5 and the Streptomyces K15 transpeptidase can be super- imposed with a root mean square deviation of 0.89 A ˚ . If so, then Glu195 in NG PBP 4 is unlikely to be suitably positioned to participate directly in catalysis. The absence of other suitable candidates for the acidic pK a in the active site, at least based on sequence alignment data, focuses attention back to Lys102 as potentially responsible for the acidic pK a . Such a low pK a for a lysine would be unusual but not unprecedented (e.g. acetoacetate decarboxylase, pK a 5.9 [40]). A similar argument also applies to NG PBP 3, and suggests Lys61 (SXXK motif) and Lys404 (KTG motif) as most likely responsible for the acidic and basic pK a values, respect- ively (Table 5). Most significantly, this assignment would suggest the same mechanistic function of the two highly conserved Lys residues in NG PBP 4 and NG PBP 3 as in EC PBP 5 [14,15] and would support a common mechanism of catalysis amongst class A and C LMM PBPs. Note added in proof: A recently reported structural and mutagenesis study of the Streptomyces K15 PBP also implicates the SXXK lysine as the catalytic base during acylation [41]. Acknowledgements Supported by NIH grants GM-60149 (WGG), AI-36901 (RAN), and GM-066861 (CD). We thank Dr Ann E. Jerse for providing valuable information and references on N. gonorrhoeae growth conditions during review of this manuscript. Table 6. Sequence alignment of amino acid residues in conserved motifs of NG PBP 3, NG PBP 4, EC PBP 5, TEM b-lactamase and Streptomyces K15 DD -transpeptidase. Conserved, mechanistically significant, or potentially mechanistically significant residues are shown in bold. Protein Motif SXXK SXN X-(like) loop (e. . .gl) KT(S)G NG PBP 3 54 VNPASTMKLVT 64 294 DMNKRSDNLIA 304 343 VLENGSGLSRK 353 399 GLLRLKTGTLN 409 NG PBP 4 95 MPIASISKLMS 105 151 LSLMSSENRAT 161 192 RFYEPTGLNFQ 202 258 NIELQKTGYIR 266 EC PBP 5 40 RDPASLTKMMT 50 105 DINLQSGNDAC 115 146 HFQTVHGLDAD 156 210 NVDGIKTGHTD 218 Streptomyces K15 31 RSTGSTTKIMT 41 91 GLMLPSGCDAA 101 138 HFDSFDGIGNG 148 208 GAIGVKTGSGP 218 b-Lactamase 66 FPMMSTFKVLL 76 125 AAITMSDNTAA 136 162 LDRWEPELNEA 172 229 WFIADKSGAGE 239 Table 5. pK a values and residue assignments for TEM b-lactamase, EC PBP 5, NG PBP 3, and NG PBP 4. NA, data not available; AA, amino acid. pK a Enzyme TEM b-Lactamase EC PBP 5 a NG PBP 3 b NG PBP 4 c pK a value AA residue pK a value AA residue pK a value AA residue pK a value AA residue pK 1 5–6.2 Glu166 d 8.2 Lys47 6.8 Lys61? 6.9 Lys102? pK 2 8.5 Lys234 e 11.1 Lys213 9.8 Lys404 10.1 Lys261 Anomalous buffers NA TEA, Carbonate TEA, Caps TEA a [15]; b [13a]; c this paper; d [35]; e [36]. 30 M. 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